A recent expansion of our understanding of the biological role and clinical utility of oligosaccharides has created a need for new synthetic methodologies for inexpensively producing large quantities of these compounds. (Lowe et al. Cell 1990, 63, 475-484; Feizi et al., Trends Biochem. Sci. 1991, 16, 84-86) .beta.-Galactosides are an important class of oligosaccharide for which many new bioactivities have recently been characterized and for which new synthetic methodologies are needed. For example, N-acetyl-D-lactosamine (LacNAc or Gal.beta.1,4GlcNAc) has been determined to have clinical utility with respect to cellular recognition processes. (Feizi et al. Biochemistry 1994, 33, 6342-6349; Liu et al., Immunol. Today 1993, 14, 486-490; Sparrow et al. J. Biol. Chem. 1987, 262, 7383-7390.) Conventional methodologies for synthesizing N-acetyl-D-lactosamine are inadequate for commercial application.
For example, conventional synthetic methodologies from organic chemistry may be employed for producing N-acetyl-D-lactosamine. (Toshima et al. Chem. Rev. 1993, 93, 150314 1531; Schmidt et al., Angew. Chem. Int. Ed. Engl. 1986, 25, 212-235; Paulsen et al., ibid. 1982, 21, 155-173; Sinay et al., Pure Appl. Chem. 1991, 63, 519-528; Paulsen et al., Angew. Chem., Int. Ed. Engl. 1990, 29, 823-839). However, these conventional organic methodologies are complex and involve many steps and undesired side reactions.
When enzymatic methodologies are available for synthesizing oligosaccharides, they are often preferred over organic methodologies. As compared to chemical methodologies, many enzymatic methodologies are characterized by their relative simplicity and absence of side reactions. For example, N-acetyl-D-lactosamine (LacNAc) may be enzymatically synthesized using either .beta.1,4-glycosyltransferase or .beta.-galactosidase.
In the .beta.1,4-glycosyltransferase-catalyzed reaction, a glycosyl group is transferred from a donor, e.g. UDP-galactose, to an acceptor saccharide to form the disaccharide N-acetyl-D-lactosamine (LacNAc). In an improved version of the .beta.1,4-glycosyltransferase-catalyzed reaction, the UDP-galactose donor is regenerated in situ. (Ichikawa et al. J. Am. Chem. Soc. 1992, 114, 9283-9298.) The process gives high regioselectivity and yield, but the limited availability of the enzymes and their high cost and instability have hampered their use in large scale synthesis (Auge et al. Carbohydr. Res. 1990, 200, 257-268).
.beta.-Galactosidase is an enzyme which, in its preferred direction, hydrolyzes glycosidic bonds, i.e., a glycosyl group is transferred from a donor glycoside or oligosaccharide to water. However, at high substrate concentrations, the reverse glycosyl transfer reaction may occur, viz., .beta.-galactosidase may be employed for catalyzing transglycosylation reactions. The advantages of the galactosidase-catalyzed reaction are the enzyme's low cost, high stability and simple reaction conditions, i.e., there is no need for sugar nucleotides. (David et al. Chemtracts-Org. Chem. 1994, 7, 92-95; Takayama et al. Bioorg. Med. Chem. Lett. in press.) There are, however, some disadvantages associated with the use of galactosidase-catalyzed reactions. Because of the hydrolytic nature of the enzyme, the yields hitherto reported have been low and the desired compound was difficult to isolate from reaction mixtures containing quite similar products. (Usui et al. Carbohydr. Res. 1993, 244, 315-323; Herrmann et al. Angew. Chem. Int. Ed. Engl. 1993, 32, 1342-1343; Herrmann et al. Tetrahedron Lett. 1993, 34, 3091-3094.)
For representative examples of other glycosidase-catalyzed synthesis, see Nilsson et al. Trends Biotechnol. 1988, 6, 256-264; Crout et al. Pure Appl. Chem. 1992, 64, 1079-1084; Lehmann et al. Carbohydr. Res. 1979, 71, 65-73; Gais et al., Zeissler et al Tetrahedron Lett. 1988, 29, 5743-5744; Sauerbrei et al., Thiem et al., Tetrahedron Lett. 1992, 33, 201-204; Kobayashi et al. J. Am. Chem. Soc. 1991, 113, 3079-3084; Lopez et al.J. Org. Chem. 1994, 59, 737-745; Petit et al. Tetrahedron Lett. 1991, 32, 6125-6128; David et al., Chemtracts-Org. Chem. 1994, 7, 92-95; Takayama et al., Shimazaki et al., Bioorg. Med. Chem. Lett. in press; Ajisaka, et al. Carbohydr. Res. 1994, 259, 103-115.
Substrate specificities can limit the applicability of enzymatic methodologies. The limitations introduced by substrate specificities may sometimes be partially overcome by chemically modifying the substrate. Accordingly, chemo-enzymatic methodologies can have an expanded synthetic scope as compared to purely enzymatic methodologies.
What is needed is a method for synthesizing .beta.-galactosides in high yield and without significant side reactions using a transglycosylation reaction catalyzed by .beta.-galactosidase.